Several lines of evidence indicate that the functional status of telomeres is mechanistically related to the growth potential of normal and cancerous cells. Preserving telomere function, via expression of ectopic telomerase, prevents genomic instability and immortalization of cancer prone mammary epithelial cells while triggering telomere dysfunction by DNA damaging agents halts breast cancer cell growth, predominantly by induction of senescence. Recognizing that the functional status of the telomere is critically linked to cellular fate, our overall objectives are to better understand mechanisms underlying the putative tumor suppressive role of telomerase and DNA damage-induced telomere dysfunction and senescence. We hypothesize that: (1) telomerase confers genomic stability via telomere length-dependent and -independent mechanisms; (2) overexpression of the telomere binding protein, TFR2, will provide for increased genomic stability even in the presence of accelerated telomere shortening; (3) both telomere breakage induced by topoisomerase II and deregulation of TRF2 contribute to telomere dysfunction and senescence in adriamycin-treated breast tumor cells; and (4) adriamycin-induced senescence is dependent on p53 recognizing telomeres as damaged DNA. We will test these possibilities using two well-characterized in vitro models of breast cancer, namely Li Fraumeni human mammary epithelial (HME) cells and MCF-7 human breast cancer cells. Our first specific aim involves using a panel of hTERT mutants to define the mechanisms by which ectopic telomerase confers genomic stability in HME cells, focusing on the possibility of a telomere length-independent "capping" event and assessing the role of telomere binding proteins in maintenance of genomic stability and overall telomere length/function. The second aim is to define the mechanisms of adriamycin-induced telomere dysfunction and senescence in breast tumor cells. Here, both cytogenetic and genetic approaches will be employed to define (1) the involvement of topoisomerase ll-mediated telomere breakage and TRF-2 deregulation in adriamycin-induced telomere dysfunction and senescence; (2) TRF2 modifications in breast tumor cells following adriamycin treatment; and (3) the p53 dependence of adriamycin-induced telomere dysfunction and senescence. The data generated will contribute in a significant way to better understanding the relationships between telomere status, genomic stability and cellular fate of human mammary epithelial cells, likely leading to the development of new preventative and therapeutic strategies for breast cancer.